Roles for TDP-43 and FUS in ALS Using Motor Neurons from Embryonic Stem Cells

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种成人起病的神经退行性疾病，其上下运动神经元过早丧失导致致命瘫痪，典型病程为一至五年。最近，两种DNA/RNA结合蛋白TAR DNA结合蛋白(TDP-43)和肉瘤融合蛋白(FUS)的突变被确定为遗传性ALS的主要原因，并可能导致对ALS发病机制的研究发生范式转变。然而，仍然需要研究这些蛋白在与ALS进展相关的细胞类型中的功能，包括运动神经元和神经胶质细胞。虽然在SOD1突变引起的遗传性ALS中，来自周围细胞的贡献已经得到很好的证实，但TDP-43或FUS突变后非细胞自主机制对疾病进展的影响是完全未知的。这项提议寻求使用来自现有转基因小鼠的胚胎干细胞来检查TDP-43或FUS在纯化运动神经元中减少或突变的后果。这些基因改变的细胞内在后果将通过诱导胚胎干细胞分化为运动神经元来确定，运动神经元缺乏或表达突变的TDP-43或FUS。与其采取候选方法在增殖细胞系中寻找已被确定为TDP-43或FUS靶点的少数基因，分离的运动神经元培养将被用来鉴定在目标1中因失去TDP-43或FUS而改变的完整RNA集，然后在目标2中询问是否有任何RNA在TDP-43或FUS突变时改变，如果是，它们是否与在丧失功能时观察到的RNA相同。通过从纯化的起始材料中采取如此全面和系统的方法，很可能会发现受影响的特定靶点和信号通路--这些发现可能会阐明疾病的潜在机制，并为未来的治疗开发提供基础。这些目标的成功将导致对ALS疾病机制的更好理解，并将为未来对神经胶质细胞类型的研究提供理论基础，以确定操作支持非神经元细胞是否会对TDP-43或FUS突变的ALS患者提供治疗益处。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which premature loss of upper and lower motor neurons leads to fatal paralysis with a typical disease course of one to five years. Mutations in two DNA/RNA binding proteins, TAR DNA-binding protein (TDP-43) and Fused in Sarcoma (FUS) have recently been identified as primary causes of inherited ALS and have led to what is likely to be a paradigm shift in efforts to understand the pathogenesis of ALS. Yet there remains a need to examine the functions of these proteins in cell types relevant for ALS progression, both motor neurons and glial cells. While a contribution from surrounding cells is well established in inherited ALS caused by SOD1 mutations, the impact of non-cell autonomous mechanisms in disease progression following mutation in TDP-43 or FUS is completely unknown. This proposal seeks to use embryonic stem cells derived from existing transgenic mice to examine the consequences of either reduction or mutation of TDP-43 or FUS in purified motor neurons. Cell intrinsic consequences of alterations in these genes will be determined by inducing differentiation of embryonic stem cells into motor neurons which either lack or express mutant TDP-43 or FUS. Rather than take a candidate approach to pursue the few genes that have already been identified as targets of TDP-43 or FUS in proliferating cell lines, isolated motor neuron cultures will be used to identify the complete set of RNAs that are altered by loss of TDP-43 or FUS in Aim 1, and then in Aim 2 to ask whether any RNAs are altered upon mutation of TDP-43 or FUS and if so, if they are the same as those observed upon loss of function. By taking such a comprehensive and systematic approach from purified starting material it is likely that the specific targets and signaling pathways affected can be uncovered - discoveries that might elucidate underlying mechanisms for disease and provide a basis for future therapeutic developments. Success in these goals will lead to greater understanding of ALS disease mechanism and will provide rationale for future studies in glial cell types to determine whether manipulation of supporting non-neuronal cells would provide therapeutic benefit in ALS patients with mutations in TDP-43 or FUS.